The specific aims of the proposed project are as follows: 1. To continue the purification of various forms of the rate-limiting enzymes involved in the synthesis of Gamma-aminobutyric (GABA), glycine and taurine, namely L-glutamate decarboxylase (GAD), serine transhydroxymethylase (serine-THM), and cysteinesulfinic and cysteic acids decarboxylase (CSAD and CAD, respectively). The purified preparations will be used as antigens for the production of specific antibodies either through the conventional method or by hybridoma technique to obtain a monoclonal antibody. 2. To identify the neuronal pathways which employ GABA, glycine or taurine as their neurotransmitter by immunocytochemical localization of their synthetic enzymes, namely GAD, serine-THM and CSAD/CAD, respectively. 3. To identify the site of synthesis, transport and storage of GAD, serine -THM and CSAD/CAD. We plan to use direct peroxidase-labeled Fab method to localize these enzymes within subcellular organelles since we have shown that peroxidase-labeled Fab can penetrate the membrane of cellular organelles. 4. To identify and isolate the factor which is responsible for the conversion of high MW GAD to low MW GAD. We also plan to elucidate the mode of action of this factor(s) with a special emphasis on its possible role in the regulation of GABA level in the nerve terminal. 5. To elucidate the interrelationship of neurons which employ GABA as transmitter and those employing glycine and taurine as transmitters using double staining techniques and a combination of autoradiography and immunocytochemistry. The possibility of coexistence of GABA and other neuropeptides in the same neuron will also be examined using the same approach. 6. To elucidate the nature of glial GAD and its relationship to neuronal GAD with a view of determining the functional role of glia in neurotransmission. The Principal Investigator has experience in all aspects of the proposed studies ranging from enzyme purification to immunochemical and immunocytochemical identification of GABA-ergic pathway (see Dr. Wu's publication and Appedix) and hence has shown the feasibility of the proposed project.